Difference between revisions of "Part:BBa K2052018"

Latest revision as of 01:17, 20 October 2016

Butyryl-CoA:acetate CoA-transferase

Usage & Biology

This basic part consists of a coding region ( ButCoAT ) and enables E.coli to secrete ButCoAT.

ButCoAT is an enzyme (Butyryl-CoA:acetate CoA-transferase) (EC 2.8.3.8) (Uniprot:C7GB37) which converts Acetyl-CoA (Acetyl-CoA is readily present in the cytosol and it can be directly converted into Butyrate) into Butyrate and can be found in Roseburia intestinalis L1-82. The equation of the reaction (acyl-CoA:acetate CoA-transferase) is given down below :

Figure 1: Butanoyl-CoA + Acetate <=> Butanoic acid + Acetyl-CoA

Figure 2: Crystal Structures of Acetobacter aceti Succinyl (Butyryl)-CoA:Acetate CoA-Transferase Reveal Specificity Determinants and Illustrate the Mechanism Used by Class I CoA-Transferases.(Mullins, E.A. et al., 2012)

Our protein coding region (ButCoaT)is digested from our whole construct with the enzymes PstI and Xbal.

Gel Results

We have loaded an uncut version of K2052015 next to an EcoRI single digested one. In the third lane ButCoat (K2052018) was loaded with its single digested version. Digested ones gave a sharp lane at 3600 bp as we expected.

Characterization

Ligated Parts Transformation Results

Figure 4. After ligating ButcoaT and RFP, we have obtain colonies 2:1 (insert:vector) ratio, and transform them into E.coli BL21.

Confirmational PCR Result

Figure 5. Constitutive promoter-RBS-ButCoaT-GFP-Double terminator cloned into pSB1C3 and after ligation we have transformed into E.coli BL21. Colony PCR results here you can see; one primer stick on insert and another stick on vector. The expected lenght of product is around 850 bp.

Flowcytometry Result

After checking with confirmational PCR we have tried to validate RFP fluoresence experimenttaly through Fluorescence Microscope, Here you can see the analysis.

Figure 6. After measuring RFP signals with channel FL3 we have obtained these graphs. Here again upper triplet stand for Mock(reference point) analysis that is the bacterial culture transformed with only ButCoaT. Middle triplet stand for only RFP expressed bacterial culture analysis which gave a peak that could be used as a control group. The bottom triplet was RFP tagged ButCoaT expressed bacterial group and as we have shown there is any signal depend on RFP activity.

Since ButCoAT was tagged with RFP, to test whether it is functional or not, we have used fluoresence microscopy technique to show its expression is properly done or not. However, because ButCoAT doesn’t have a stop codon at the end it fused with RFP and they both become unfunctional from this fusion. Therefore, we could not see any peak.

Modelling

Molecule versus second

Figure 7.The figure above shows the increase in ButCoAT molecules over a time span of 10 seconds.

Molecule versus second

Figure 8.The figure above shows the molar increase in Butyrate concentration over a time span of 10 seconds.

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2052018 short</partinfo>
-ButCoAT is an enzyme (Butyryl-CoA:acetate CoA-transferase) (EC 2.8.3.8) (Uniprot:C7GB37) which converts Acetyl-CoA (Acetyl-CoA is readily present in the cytosol and it can be directly converted into Butyrate.) into Butyrate and can be found in Roseburia intestinalis L1-82. The equation of the reaction (acyl-CoA:acetate CoA-transferase) (KEGG Reaction No. : R01179)  is given down below :
-[[File:|none|500px|]]
+==Usage & Biology==
-Butanoyl-CoA + Acetate <=> Butanoic acid + Acetyl-CoA
-[[File:ButCoaTStructure.gif|none|frame|500px]]
+This basic part consists of a coding region ( ButCoAT ) and enables  E.coli to secrete ButCoAT.
-Figure 1: Crystal Structures of Acetobacter aceti Succinyl(Butyryl)-CoA:Acetate CoA-Transferase Reveal Specificity Determinants and Illustrate the Mechanism Used by Class I CoA-Transferases.(Mullins, E.A. et al., 2012)
-===Butyrate===
+ButCoAT is an enzyme (Butyryl-CoA:acetate CoA-transferase) (EC 2.8.3.8) (Uniprot:C7GB37) which converts Acetyl-CoA (Acetyl-CoA is readily present in the cytosol and it can be directly converted into Butyrate) into Butyrate and can be found in Roseburia intestinalis L1-82. The equation of the reaction (acyl-CoA:acetate CoA-transferase) is given down below :
-Butyrate is a short chain fatty acid which is formed by bacterial fermentation of carbohydrates (Sengupta et al., 2005).
-Butyrate putatively suppresses colorectal cancer (CRC). It induces apoptosis and differentiation, inhibits proliferation of tumorous cells in colon flora. At the intestinal level, butyrate plays an administrative part on the transepithelial liquid transport, improves mucosal aggravation and oxidative status, strengthens the epithelial protection obstruction, and regulates instinctive affectability and intestinal motility.
+[[File:Pathwayson.png|none|frame|700px|]]
-==Modeling==
+Figure 1: Butanoyl-CoA + Acetate <=> Butanoic acid + Acetyl-CoA
-[[File:ButCoaTModeling.jpeg|none|700px|caption]]
-The figure above shows the increase in ButCoAT molecules over a time span of 10 seconds.
-[[File:ButyrateModeling.jpeg|none|700px|caption]]
-The figure above shows the molar increase in Butyrate concentration over a time span of 10 seconds.
-==Design Notes==
-E.coli is synthesized suitable for Codon Bias.
+[[File:ButCoaTStructure.gif|none|center|frame|500px]]
+Figure 2: Crystal Structures of Acetobacter aceti Succinyl (Butyryl)-CoA:Acetate CoA-Transferase Reveal Specificity Determinants and Illustrate the Mechanism Used by Class I CoA-Transferases.(Mullins, E.A. et al., 2012)
-In the expression of pet28a (expression vector) , stop codon is removed for the addition of  HisTag.
+Our protein coding region (ButCoaT)is digested from our whole construct with the enzymes PstI and Xbal.
-==DNA Gel Analysis==
-===Characterization===
-PCR Conformation of ButCoAT + pSB1C3
-[[File:ButCoaTGelImage.jpeg|none|500px|]]
-We have loaded the uncut version of K2052015 (RBS + ButCoAT) next to EcoRI single digested one. In the third lane, RBS ligated ButCoAT (K2052018) was loaded with its single digested version. Digested ones gave sharp lane at 3600 bp as we expected.
-==Source==
+===Gel Results===
-Roseburia intestinalis L1-82
+[[File:METU HS DENEME688889.jpeg|500px]]
+We have loaded an uncut version of K2052015 next to an EcoRI single digested one. In the third lane ButCoat (K2052018) was loaded with its single digested version. Digested ones gave a sharp lane at 3600 bp as we expected.
-<!-- -->
+==Characterization==
-<span class='h3bb'>Sequence and Features</span>
-<partinfo>BBa_K2052018 SequenceAndFeatures</partinfo>
-<!-- Uncomment this to enable Functional Parameter display
+===Ligated Parts Transformation Results===
-===Functional Parameters===
-<partinfo>BBa_K2052018 parameters</partinfo>
-<!-- -->
-==References==
-Weinstock G., Sodergren E., Clifton S., Fulton L., Fulton B., Courtney L., Fronick C., Harrison M., Strong C., Farmer C., Delahaunty K., Markovic C., Hall O., Minx P., Tomlinson C., Mitreva M., Nelson J., Hou S.  Wilson R.K. (2009 , August ). UniProtKB - C7GB37 (C7GB37_9FIRM). Retrieved from http://www.uniprot.org/uniprot/C7GB37?sort=score
-Sengupta, S., Muir, G.J., Gibson, P. R. (2005). Does Butyrate Protect from Colorectal Cancer?
-https://swissmodel.expasy.org/templates/4eu8.1
+[[File:METU HS DENEME9.jpeg|center|250px]]
+Figure 4. After ligating ButcoaT and RFP, we have  obtain colonies 2:1 (insert:vector) ratio, and transform them into E.coli BL21.
+===Confirmational PCR Result===
+[[File:METU_HS_DENEME6888.jpeg|center|500px]]
+Figure 5. Constitutive promoter-RBS-ButCoaT-GFP-Double terminator cloned into pSB1C3 and after ligation we have transformed into E.coli BL21. Colony PCR results here you can see; one primer stick on insert and another stick on vector. The expected  lenght of product is around 850 bp.
+==Flowcytometry Result==
+After checking with confirmational PCR we have tried to validate RFP fluoresence experimenttaly through Fluorescence Microscope, Here you can see the analysis.
+[[File:METU_HS_DENEME3luolan.jpeg|center|800px]]
+Figure 6. After measuring RFP signals with channel FL3 we have obtained these graphs. Here again upper triplet stand for Mock(reference point) analysis that is the bacterial culture transformed with only ButCoaT. Middle triplet stand  for only RFP expressed bacterial culture analysis which gave a peak that could be used as a control group. The bottom triplet was RFP tagged ButCoaT expressed bacterial group and as we have shown there is any signal depend on RFP activity.
+Since ButCoAT was tagged with RFP, to test whether it is functional or not, we have used fluoresence microscopy technique to show its expression is properly done or not. However, because ButCoAT doesn’t have a stop codon at the end it fused  with RFP and they both become unfunctional from this fusion. Therefore,  we could not see any peak.
+==Modelling==
+Molecule versus second
+[[File:ButCoAT_graph.jpeg|900px]]
+Figure 7.The figure above shows the increase in ButCoAT molecules over a time span of 10 seconds.
+Molecule versus second
+[[File:Butyrate graph.jpeg|900px]]
+Figure 8.The figure above shows the molar increase in Butyrate concentration over a time span of 10 seconds.